CRISPR/Cas9 System as a Valuable Genome Editing Tool for Wine Yeasts with Application to Decrease Urea Production.

Area of Science:

• Yeast genetics and metabolism
• Molecular biology and genetic engineering
• Enology and wine fermentation

Background:

• Laboratory strains of Saccharomyces cerevisiae have extensive genetic tools, but polyploid industrial yeast metabolism remains poorly understood.
• Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) engineering are revolutionizing genetic modification in industrial yeasts.

Purpose of the Study:

• To apply the CRISPR/Cas9 system for genetic engineering in commercial Saccharomyces cerevisiae wine strains.
• To generate yeast strains with reduced urea production by targeting the CAN1 arginine permease pathway.

Main Methods:

• CRISPR/Cas9 gene editing was employed to eliminate the CAN1 pathway in two commercial S. cerevisiae strains (EC1118, AWRI796).
• The engineered strains were tested in a wine-model environment using Chardonnay and Cabernet Sauvignon grape musts.
• Urea production and fermentation efficiency were monitored for both wild-type and mutant strains.

Main Results:

• CRISPR/Cas9 successfully generated S. cerevisiae mutants with impaired arginine metabolism.
• Engineered EC1118 and AWRI796 strains showed reduced urea production by 18.5% and 35.5%, respectively.
• Both wild-type and mutant strains completed must fermentation within 8-12 days, indicating retained fermentative capacity.

Conclusions:

• The CRISPR/Cas9 system is effectively established in S. cerevisiae wine yeasts for targeted genetic modification.
• Genetic engineering of the CAN1 pathway successfully reduced urea production in a wine-model environment.
• This demonstrates the potential for improving wine quality through precise yeast strain engineering.